The Ton system is composed of proteins ExbB, ExbD and TonB, which form a complex in the inner membrane (Lazdunski et al 1998). The tonB gene is located at 28 min on the E.coli chromosome and the exbB and exbD genes form an operon at 65 min (Lazdunski et al 1998). The physiological role of the Ton system is transduction of energy from the pmf of the cytoplasmic membrane into the periplasm (Larsen et al 2001).
1.9.2.1. Arrangement of the Ton system
TonB and ExbD are anchored by their N-terminal end to the cytoplasmic membrane and extend into the periplasm (Postle and Skare 1988, Kampfenkel and Braun 1992). ExbB is an integral cytoplasmic membrane protein with three transmembrane
segments (Fischer et al 1989). ExbB, ExbD and TonB form a complex in the inner membrane via their transmembrane regions with ExbB at the centre of the complex (Lazdunski et al 1998). However, the stoichiometry may not be 1:1:1 as TonB may dimerise, and ExbB is thought to be expressed in greater quantities than ExbD and TonB (Chang et al 2001, Fischer et al 1989).
1.9.2.2. Similarities of the Ton and Tol systems
ExbB and ExbD are functionally and structurally homologous to TolQ and TolR respectively and it is thought that TolQR can complete the role of ExbBD and vice versa (Eick-Helmerich and Braun 1989, Braun and Hermann 1993). However, TolA and TonB are not interchangeable although when the N-terminal transmembrane region of TolA was exchanged with the same region of TonB, the mutated protein exhibited TonB activity, consistent with the sequence similarity observed in this region (Braun and Hermann 1993).
1.9.2.3. TonB box
The N-terminal domain of TonB interacts with outer membrane receptors (Brewer et
al 1990).
and Kadner 1999). The crystal structure of BtuB shows that the TonB box is located in the plug domain and that the TonB box shifts in position, on binding of vitamin B12 (Chimento et al 2003). This supports the hypothesis that the interaction of TonB with the TonB box is involved in the removal of the ligand from the binding site of the receptor (Cadieux et al 2000).
The TonB box has been identified in a number of different colicins, suggesting that Ton-dependent colicins could interact with TonB in a similar way to the Ton- dependent receptors (Wiener et al 1997). Mutagenesis studies have shown that colicin M requires the TonB box of FhuA as well as its own TonB box for uptake (Schöffler and Braun 1989). Therefore, the receptor TonB box-TonB interaction may induce the conformational change required for the colicin to enter the periplasm and then the colicin may interact with TonB independently.
1.9.2.4. Model for Ton-dependent translocation of colicins
Two models have been proposed to explain how TonB energy transduction can cause translocation.
The propeller model (Chang et al 2001) proposes that TonB remains associated with the cytoplasmic membrane with a rotation of its carboxy terminus, initiated by ExbB, ExbD and the pmf. When this propeller of TonB binds to the TonB-dependent
receptor, the rotary motion causes the receptor to release its ligand into the periplasm. This model was based on the observation that the carboxy-terminal domain of TonB is a rigid, strand-exchanged dimer, likened to a propeller, but there is currently very little data to support the model (Postle and Kadner 2003).
The shuttle model (Letain and Postle 1997, see Figure 1.13) proposes that TonB starts in a complex with ExbB and ExbD in an unenergised state. ExbB and ExbD then use the cytoplasmic membrane pmf to convert TonB into an energised state. The C- terminal domain of TonB would then contact the outer membrane, whilst remaining in contact with the cytoplasmic membrane. This then causes the N-terminal domain of TonB to be released from ExbB and ExbD so it could interact with a Ton-dependent receptor in the outer membrane. This interaction would cause release of
conformationally stored potential energy from TonB and release of the ligand from the receptor into the periplasm. Current evidence, including the localisation of TonB at both the outer and cytoplasmic membranes and in soluble intermediate forms,
Figure 1.13 The shuttle model for translocation of a ligand across the periplasm via TonB- dependent energy transduction (from Postle and Kadner 2003). 1. TonB is associated with ExbD and ExbB at the cytoplasmic membrane. 2. ExbB, ExbD use the cyptoplasmic membrane pmf to energise TonB. 3. TonB associates with the outer membrane, initially with non-
transporter proteins such as Lpp. The binding of ligand to FepA induces a conformational change allowing the receptor to interact with TonB causing a release of conformationally stored potential energy and release of ligand into the periplasm. 4. The energetically uncharged TonB then shuttles back to the cytoplasmic membrane.
It is as yet unknown whether colicins could use a similar mode of translocation to the natural ligands of Ton-dependent receptors. A model for the translocation of colicin Ia has been proposed, based on the crystal structure of this colicin (Wiener et al 1997). In this model, the TonB box in the translocation domain of the colicin competes with the TonB box of Cir, the Ton-dependent receptor for colicin Ia, for binding to TonB. The displacement of TonB from the receptor, allows colicin Ia to move across the outer membrane, into the periplasm (see Figure 1.14).
TonB ExbB ExbD Periplasm Outer membrane Inner membrane Receptor TonB box Receptor Receptor-binding domain Translocation domain (containing TonB box)
Cytotoxic domain Linker region TonB ExbB ExbD Periplasm Outer membrane Inner membrane Receptor TonB box Receptor Receptor-binding domain Translocation domain (containing TonB box)
Cytotoxic domain
Linker region
Figure 1.14 Proposed mechanism for translocation of Ton-dependent colicins (adapted from Wiener et al 1997). On binding of the receptor-binding domain of the colicin to the Ton-
dependent receptor, the TonB box of the colicin competes with the TonB box of the receptor for binding to TonB. This would then allow the cytotoxic portion of the colicin to enter the